Most lymphocytes have a lifespan shorter than their host, and dying cells must be continually replaced to maintain homeostasis. Most T lymphocytes are produced in the thymus. However, unlike most other systems of steady-state differentiation, the thymus does not contain stem cells. Rather, low numbers of precursors are constantly supplied via the bloodstream to the thymus, where they are induced to develop into functional T cells. The later stages of thymocyte development are mainly dictated by the T cell receptor (TCR). However, the early stages of differentiation (defined by the absence of CD4/8 lineage markers) constitute the longest period of intrathymic development, and the largest number of cell divisions. In addition, T lineage commitment and alpha/beta vs. gamma/delta lineage divergence also occur at this stage. Since these early cells lack TCR expression, other factors must control their differentiation and proliferation. However, the nature of this process is largely unknown. Data provided in this application show that CD4-8- precursors undergo a precise pattern of tissue translocation during differentiation. The earliest precursors are found deep in the cortex near the medulla (cortico-medullary junction, CMJ), consistent with the location of vessels that permit extravasation from the blood. These precursors then migrate specifically in the direction the capsule, undergoing several develomental stages and completely traversing the cortex before accumulating in the sub-capsular zone (SCZ). In the SCZ, CD4-8- precursors differentiate to the CD4+8+ stage, after which the polarity of migration is reversed, and cells move back across the cortex. During this transmigration, early thymocytes sequentially encounter stratified stromal compartments formed during embryogenesis. The hypothesis put forth in this application is that each sequential microenvironment provides a unique combination of differentiative and proliferative stimuli, and that development is induced, and homeostasis controlled, by regulating the movement of precursors through these environments. The aims of this application are to define the developmental events that occur within each anatomic zone, and to deterrnine the factors that induce and/or facilitate cell migration and polarity in these regions. In addition to promoting a better understanding of these basic processes, the proposed studies are expected to facilitate longer term goals of defining the differentiative and proliferative stimuli contained within each compartment and consequently that control the process of T cell development.